Introduction

In a developing country like India, poultry serves as one of the means of satisfying the increased demand for animal protein. Presently, chicken meat is on demand as a cheap source of protein with low cholesterol value. Therefore adaptation of broiler farming is increasing day by day by farmers. Antibiotics have long been used as growth promoters. Now a day due to residual effect of antibiotics on human health the use of many antibiotics in food production is banned or going to be banned. Antibiotic free food products are of greater concern for health conscious consumers in India. Considering these facts in mind the feeding of non-antibiotic growth promoters such as prebiotics, probiotics, and synbiotics finds a potential substitute for antibiotics. This study was therefore conducted to evaluate the effect of prebiotic, probiotic and synbiotic on serum biochemical and immunological status of Vencob broiler chickens in hot climatic conditions of Odisha.

Materials and Methods

A total of 360 numbers of day-old Vencob broiler chicks of either sex were procured from the Eastern Hatcheries Pvt. Ltd., Bhubaneswar. The average maximum and minimum ambient temperature during the six weeks of experimental period ranged from 37.2 ^oC to 41.3 ^oC (average 39.25 ^oC) and 21.7 ^oC to 24.8 ^oC (average 23.8 ^oC), respectively. There were 12 pens, each having a floor area of 40 sq. feet i.e. 8 feet x 5feet. The chicks were wing banded, weighed and randomly distributed into four dietary treatment groups. Each group was again divided into three replicates having thirty chicks in each replicate pen (Table 1).

Table 1: Details of experimental diets

Fresh rice husk was used as litter material. The chicks were brooded by using incandescent electric bulbs. During brooding, adequate light of 24 hrs and ventilation were facilitated. Cleaned and disinfected feeders and waterers were provided in the pen as per the requirements of the birds. Fresh clean water was provided twice daily in clean waterers inside the pen. Feed and water were provided ad libitum. All the chicks were vaccinated against Ranikhet disease (B1 Strain, Ventri®) on 7th and 21st day of age and Infectious Bursal Disease (Live Vaccine Intermediate Standard Strain Ventri®) on 14th and 35th day of age. Routine managemental practices were adopted for all treatment groups as per standard practices.

The starter and finisher rations prepared in ICAR- Central Institute for Women in Agriculture (ICAR-CIWA), Bhubaneswar were fed to chicks during the experimental period. The dietary treatments were: 1 – Control; 2 – Basal diet supplemented with prebiotic (400 g per tonne of starter as well as finisher diets); 3 – Basal diet supplemented with probiotic (100 and 50 g per tonne of starter and finisher diets, respectively); 4 – Basal diet supplemented with synbiotic (500 g per tonne of starter as well as finisher diet). The composition and dose rate of prebiotic, probiotic and synbiotic used in the diet have been presented in Table 2.

Table 2: Composition of Prebiotic/ Probiotic/ Synbiotic used in the diet

The chicks were fed with starter ration up to 21 days and finisher ration from 22 to 42 days of age as per BIS (1992) recommendations. The chicks under treatment were provided with dietary supplemented ration from day old to 42nd day of age. During the period of study (0-6 weeks) all the birds were provided with starter diet (with 3005 kcal of ME/kg of ration and 22.37% CP) from 0-3 weeks of age and finisher diet (with 3120 kcal of ME/kg of ration and 20.21% CP) from 4-6 weeks of age with ad lib provision of water. Blood samples were collected from 2 birds of each replicate on 42nd day of the experiment. About 2-3 ml of blood was collected from the wing vein, present on the ventral surface of the humeral radio-ulnar joint directly beneath the skin and blood samples were collected in sterilized dry centrifuge tubes without any anticoagulant. After 2 hours the tubes were kept in incubator at 37 ^oC for 30 minutes and then centrifuged at 3000 rpm for 15 minutes to separate serum. The separated serum samples were preserved at -10 ºC for further analysis. The serum biochemical profiles i.e. total protein, albumin, globulin and Albumin: Globulin ratio, total cholesterol and triglyceride concentration, serum calcium and phosphorus levels were estimated by following the procedures described in the reagent kit supplied by Crest Biosystems, a division of Coral Clinical Systems, Goa, India.

The cellular immune response was assessed by cutaneous basophilic hypersensitivity (CBH) test in vivo by using PHA-P (Phyto-haemagglutinin phosphate). On 35th day of age, 2 birds from each replicate of each treatment were taken for CBH response. About 10 mg of PHA-P was dissolved in 10 ml of normal saline solution. Doses of 100 µg of PHA-P in 0.1 ml of normal saline were injected intradermally in the right foot pad. The normal thickness of the right foot pad and 24 hrs of post injection were measured by using a digital slide caliper (Mitituyo, Japan). CBH response were calculated by dividing post injection thickness of right foot pad by pre injection thickness of right foot pad and multiplying it by hundred.

The humoral immune response was measured by the antibody production in response to sheep red blood corpuscles (SRBC). About 0.1 ml of SRBC (0.5%) was injected into the wing vein of each bird. After five days of post inoculation, blood samples were collected from the SRBC injected birds and antibody titre was determined by HA titre methods as per Siegel and Gross (1980) and Shyam Sunder et al. (2008). The titre was expressed as log2. The data obtained in this study were analysed statistically in SPSS software (version 16.0) as per the methods outlined by Snedecor and Cochran (1994). The experiment was carried out according to the national regulations on animal welfare and with the approval of Institutional Animal Ethical Committee.

Results and Discussion

Serum Biochemical Profile

The serum biochemical profile of Vencob commercial broilers at 6 weeks of age are presented in Table 3.

Table 3: Serum biochemical profiles of broiler chicken under different treatments at 6 weeks of age

Means bearing different superscripts in the same row differ significantly (** P<0.01,* P<0.05)

The serum total protein content in the blood of broilers after 6 weeks of age ranged from 3.38 g/dl (T4) to 4.02 g/dl (T3) under different treatments. The present value is in agreement with the value of Abdel-Raheem and Abd-Allah (2011) who reported the total protein in the range of 3.26 to 3.90 g/dl in broilers. There was no significant difference in the serum protein concentration between different treatments at 6 weeks of age. Our findings is not in agreement with the report of Shareef and Al-Dabbagh (2009) who reported that addition of yeast at a rate of 1, 1.5 and 2% significantly (P<0.05) increased the level of serum total protein compared with other treatments. The effect of dietary supplementation of prebiotic, probiotic and synbiotic in the diet of broiler chicken was found to be non- significant with respect to Total protein, albumin, globulin and A/G ratio (Table 3) under different treatments.

The calcium content in the serum of broiler chicken ranged from 7.24 to 11.24 mg/dl. The calcium content was significantly higher in synbiotic group (11.24±0.08 mg/dl), probiotic group (10.57±1.59) and prebiotic group (9.02±0.2 compared to control (7.24±0.15) but the differences in between treatments were found to be non-significant. The serum phosphorus concentration ranged from 5.42 to 6.89 mg/dl at 6 weeks of age in commercial broilers. The serum calcium and phosphorus levels were numerically higher in probiotic and synbiotic group in comparison to control which is in agreement with Abdel-Raheem and Abd-Allah (2011). The positive correlation between calcium and phosphorus retention indicates that these nutrients are absorbed in a similar pattern and that there is an interrelationship in their utilization.

The serum total cholesterol concentrations (Fig. 1) was significantly reduced by dietary treatments compared to the control group (P<0.05).

Fig.1: Serum biochemicals of broiler chicken

The total cholesterol content was significantly (P<0.05) highest in control (155.33 mg/dl) compared to T2 (117.09 mg/dl), T3 (122.97 mg/dl) and T4 (112.43 mg/dl). Similarly, the serum triglycerides concentrations were significantly reduced by dietary treatments compared to the control group. The total triglycerides content was significantly (P<0.05) highest in T1 control (144.41 mg/dl) compared to T2 prebiotic (93.59 mg/dl), T3 probiotic (97.74 mg/dl) and T4 synbiotic (103.48 mg/dl) but the differences were found to be non-significant between treatment groups (T2, T3 and T4). The lower level of serum triglyceride might be due to increased level of lactic acid producing bacteria in the gut of broiler chicken and similar cholesterol depressing effect due to probiotic supplementation in broiler chicken was observed by Joy and Samuel (1997). In some study probiotic supplementation reduces the serum LDL cholesterol (Kalavathy et al., 2003) and triglycerides (Santos et al., 1995) in broiler chicken. The present finding indicates that feeding of probiotic has a cholesterol depressing effect in broiler chicken which might be due to utilization of the cholesterol present in the gastrointestinal tract by the micro-organisms present in the probiotic preparation for their own metabolism, thus reducing absorption of cholesterol as per Nelson and Gilland (1984) and Mohan et al. (1995). Further, lactobacillus which has a high bile salt hydrolytic activity is responsible for deconjugation of bile salts as per Sarono (2003). Deconjugated bile is less soluble at low pH and less absorption in the intestine and is more likely to be excreted in faeces as per Klaver and Van der mee (1993). In addition probiotic micro-organisms inhibits hydroxymethyl-glutaryl-coenzyme-A an enzyme involved in the cholesterol synthesis as stated by Fukashima and Nakon, (1995). The most important mechanism by which prebiotic eliminates cholesterol would likely be through reducing lipid absorption in intestine by binding bile acids, which results in increased cholesterol elimination and hepatic synthesis of new bile acid as described by Zhang et al. (2003). On the other hand Salma et al. (2007) have shown that cholesterol concentration in thigh and breast muscle of broilers fed with probiotics had a positive correlation with the change of the cholesterol contents in serum. Thus, it is expected that with decrease of serum cholesterol the amount of meat cholesterol is tending to decrease too.

Immunological Profile

The level of primary antibody in response to sheep red blood cells (SRBC) and cutaneous basophilic hypersensitivity (CBH) response of different treatments are presented in Table 4 and (Fig. 2 and 3).

Table 4: Level of primary antibody in response to SRBC and CBH response of different treatments

Means bearing different superscripts in the same row differ significantly (* P<0.05)

The level of primary antibody was significantly higher in T3, probiotic group (7.33±0.33) compared to T1, control group (3.67±0.67) and T2, prebiotic group (4.33±0.67) but the differences between probiotic and synbiotic group was found to be non-significant. The data indicates that birds of probiotic group are better responder to humoral antibody which might be due to stimulation of the immune system by increasing production of immunoglobulins, increased activity of macrophages and lymphocytes and stimulating the production of µ-interferon as per Haghighi et al. (2005). The current finding is in agreement with Panda et al. (2000) who reported that supplementation of probiotics along with injection of SRBC, revealed higher levels of antibody production in broiler chicks.

Fig.2: Level of primary antibody response to SRBC

Fig.3: Cutaneous basophillic hypersensitivity response in broiler chicken

On the other hand Kiewicz and Korelski (2007) reported that mannan-oligosaccharides (MOS) affects the growth of IgM in blood serum, increases the intensity of T lymphocytes proliferation and thereby increases the antibody titre towards infectious bursal diseases virus. The cellular immune response assessed by cutaneous basophilic hypersensitivity (CBH) test was significantly higher in probiotic group (215.11±2.08 mm) compared to control (200.75±2.92 mm). There was no significant difference between prebiotic, probiotic and synbiotic group. Further, there was no significant difference between prebiotic group compared to control. The present finding is in agreement with several authors like Matsuzaki et al. (2000); Haghighi et al. (2005); Mathivanan et al. (2007) and Nayebpor et al. (2007) who reported potential effect of probiotic on immunomodulation in chicken.

Conclusion

Biological experiment conducted showed significant effect of growth promoters on serum calcium and phosphorus concentration in the present study. Significantly lower serum cholesterol and triglycerides in dietary supplemented group of broilers might reduce the amount of meat cholesterol which may help in reducing the occurrence of cardiovascular heart diseases in consumers. Significantly higher antibody titre in response to sheep RBC and higher cutaneous basophilic hypersensitivity (CBH) response in dietary supplemented group is suggestive of better immunomodulatory effect. In conclusion, non-antibiotic growth promoters might be promising alternative for antibiotic growth promoters in livestock and poultry feed.

Acknowledgement

The authors are grateful to the Dean, College of Veterinary Science and Animal Husbandry, C.V.Sc & A.H, Bhubaneswar; Vice Chancellor, Orissa University of Agriculture & Technology, Bhubaneswar and Director, ICAR-Central Institute for Women in Agriculture (ICAR-CIWA), Bhubaneswar for providing the facilities and financial support to this study.

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